Figure 3.

A long-term memory trace forms in the axons of the γ MB neurons after spaced forward conditioning. A, Response to the CS+ and CS− odors 24 h after conditioning. A significant increase in %ΔF/F₀ was detected in the axons of γ MB neurons using Oct as the test stimulus after 5× spaced forward conditioning with Oct as the CS+ and Ben as the CS− compared with any other group (Kruskal–Wallis statistic of 25.561, p = 0.0009; Mann–Whitney pairwise comparisons, p ≤ 0.011). The response magnitudes for all other groups (1× forward and backward, 5× massed forward and backward, and 5× spaced backward) were similar to each other and naive animals presented with odor (see Fig. 1C) (Kruskal–Wallis statistic of 2.557, p = 0.7679; Mann–Whitney pairwise comparisons, p ≥ 0.1069). No significant differences in the %ΔF/F₀ response to the CS− (Ben) were detected among any of the conditioned groups (Kruskal–Wallis statistic of 3.11, p = 0.6831; Mann–Whitney pairwise comparisons, p ≥ 0.1926; n = 12–20 for all groups). Error bars are the SEM. **p < 0.01. B, Group time course for the response to the CS+ of Oct in the axons of γ MB neurons after spaced forward conditioning compared with spaced backward conditioning. The graph was made using the data from the same flies used for the bar graph in A. Error bars are the SEM. C, Calcium responses in the axons of γ MB neurons in animals conditioned with Ben as the CS+ and Oct as the CS−. A significant increase in %ΔF/F₀ was detected in the axons of γ MB neurons using Ben as the test stimulus after 5× spaced forward conditioning with Ben as the CS+ and Oct as the CS− compared with any other group (Kruskal–Wallis statistic of 18.02, p = 0.0029; Mann–Whitney pairwise comparisons, p ≤ 0.0045). The response magnitudes for all other groups (1× forward and backward, 5× massed forward and backward, and 5× spaced backward) were similar to each other and naive animals presented with odor (see Fig. 1C) (Kruskal–Wallis statistic of 3.799, p = 0.5787; Mann–Whitney pairwise comparisons, p ≥ 0.3284). No significant differences in the %ΔF/F₀ response to the CS− (Oct) were detected among any of the conditioned groups (Kruskal–Wallis statistic of 7.599, p = 0.1798; Mann–Whitney pairwise comparisons, p ≥ 0.0914), except for the response between the 5× massed forward and 5× spaced forward (Mann–Whitney test, p = 0.0346). n = 8–11 for all groups. Error bars are the SEM. ***p < 0.0001. D, Images of the basal fluorescence of Uas–G-CaMP expressed with 1471–Gal4 in the axons of γ MB neurons (left column). The change in fluorescence (%ΔF/F₀) that occurs after exposure to the CS+ or CS− odor is illustrated as a false color image (middle and right columns, respectively). A robust increase in calcium influx was detected in the γ axons after CS+ odor stimulation 24 h after 5× spaced forward conditioning for both odor combinations, whereas the calcium responses to the CS+ after spaced backward conditioning and to the CS− for both spaced forward and spaced backward conditioning was similar to the odor responses of naive animals.